Although of great importance in the treatment of heart failure, the cardiotonic steroids are very toxic. The toxicity is believed to be due to inhibition of the NaK-ATPase. Our final aim in these studies is to elucidate the toxicity of digitalis. Our previous studies have shown that the cardiac aglycone-NaK-ATPase complex is a mixture of at least two types of complexes, one of which dissociates eight times faster than the other. In our first step to elucidate the complexity of this drug-enzyme complex in the Na ion-Mg ions-ATPase system, we have confirmed by the new "ouabain-trapping method" that the main phosphointermediates in the Na ion-Mg ions-ATP system, the ADP-sensitive, K ion-insensitive E1P, and the K ion-sensitive, ADP-insensitive E2P are interconvertible, and that E2P can bind directly to ouabain. We will expand this study to each transient step in the ouabain binding as follows, using a highly purified eel NaK-ATPase: (1) To identify the active intermediates for ouabain binding in the various pH ranges or ligand conditions using the "ouabain-trapping method. (2) To establish the conditions for the formation of active intermediates for ouabain binding, including the sidedness-effects. In this approach, we will examine not only the membrane fragments but also the reconstituted NaK-ATPase liposomes. Such an approach will clarify the sidedness-effects on the ouabain binding and on the phosphorylation of the Nak-ATPase. (3) To investigate the transient interactions between the active intermediate and cardiotonic steroid by fluorescent changes in the binding by anthroyl derivatives of digitoxigenin and ouabain. (4) To study the phosphorylated ouabain-enzyme complex, which is formed in the initial stage of the ouabain biding, but has never been studied. We would like to characterize this complex by its binding to Na ion and K ion and by the change in its stability.